Project Abstract Acute infectious diarrhea is a worldwide problem, especially among infants, young children and immune-compromised patients. Sadly, 1.3 million children die each year, not because of the infections causing diarrhea, but due to the associated dehydration. The pro-absorptive Oral Rehydration Solution (ORS) is the only recommended oral therapy for children with acute diarrhea, yet it neither reduces diarrhea/secretion nor alters the overly active enteric nerve (ENS) activity, gut inflammation and dysbiosis ? key contributors to diarrhea. The overarching question of this application is the following: can we develop a novel anti- diarrheal therapy that is as simple and has both pro-absorptive and anti-secretory properties while reducing ENS activity, inflammation and microbial imbalance? Based upon preliminary data, Medosome Biotec, LLC and its research partners at University of Florida hypothesize that the intestinal Calcium-Sensing Receptor (CaSR) is a likely candidate target for developing such a therapeutic. The preliminary data show that CaSR is expressed in the gut epithelium, both in the absorbing surface epithelium and the secreting crypts. Also, CaSR is densely present in the ENS, both in the fluid-modulating submucosal plexus and the motility- modulating myenteric plexus. Using in vitro or ex vivo models, it was observed that activating the CaSRs either by nutritional agonists (e.g., calcium, spermine) or by chemical agonists (e.g., R568), reversed cholera toxin- induced fluid movement from secretion to absorption and reduced gut motility, inflammation and dysbiosis. Furthermore, mice lacking the CaSR displayed increased intestinal permeability, imbalanced microbiota, and skewed immune responses from regulatory towards pro inflammatory. This Phase I STTR project will use two mouse models of diarrhea [i.e., cholera toxin model of secretory diarrhea and citrobacter model of infectious diarrhea] to further examine diarrhea-therapeutic potentials of CaSR-activating nutrients/agonists, calcium (orthosteric agonist), spermine, and tryptophan (both allosteric and biased agonists). Aim 1 will examine efficacy and safety of increasing doses of calcium to define an optimal dose that produces maximal therapeutic and minimal adverse effects. Aim 2 will determine if adding spermine and/or tryptophan to calcium adds additional efficacy and safety in treating diarrhea and dysbiosis. The results from these studies will determine which strategy provides the best therapy for diarrhea: single nutrient therapy vs. combination therapy. Once an optimal recipe (composition and doses) of the CaSR antidiarrheal therapy is defined, it will be further tested in humans (Phase II/III studies). The overall goal of the STTR project is to develop a novel widely available ?child-friendly? anti-diarrheal therapy to complement current ORS for acute diarrhea. If these studies are promising, CaSR-based therapies will have clinical utility in hundreds of millions of infants and children worldwide.